At birth the human fetus switches from a glucose-dominated to a lipid-dominated energy supply since fat, or more specifically triglycerides (TG), that constitutes half of the total energy in human milk and most infant formulae, serves as the dominating energy substrate for newborn infants. Therefore, efficient digestion and absorption of dietary TG is crucial to infant growth and development.
In adults, colipase-dependent pancreatic lipase (PTL) is the main enzyme responsible for the digestion of dietary TG. In the newborn infant, and particularly in the preterm infant, exocrine pancreatic functions are not fully developed (Manson & Weaver, 1997; Arch Dis Child Fetal Neonatal Ed, 76: 206-211). Hence, in the infant, expression of pancreatic lipases is low compared to adult pancreas (Lombardo, 2001; Biochim Biophys Acta, 1533: 1-28; Li et al 1007; Pediatr Res, 62: 537-541), the intraluminal PTL activity during established fat digestion is much lower compared to adults (Fredrikzon et al, 1978; Paediatr Res, 12: 138-140) and fat malabsorption is not uncommon (Carnielli et al, 1998; Am J Clin Nutr 67: 97-103; Chappell et al, 1986; J Pediatr, 108: 439-443). In the breastfed infant, low PTL activity is compensated for by a broad-specificity lipase, bile-salt-stimulated lipase (BSSL) (EC 3.1.1.13), which is secreted both from the lactating mammary gland into the milk and from the exocrine pancreas. In preterm infants, the milk seems to provide the major part of BSSL in duodenal content during a breast milk meal (Fredrikzon et al, 1978).
Whereas fat absorption is an efficient process in healthy human adults with less than 5% of the dietary lipids excreted with the stool (Carey & Hernell, 1992, Semin Gastrointest Dis, 3: 189-208), as much as 20-30% (or more) of the dietary fat may be excreted in preterm infants for no reason other than immaturity. It is of note, however, that the extent of fat malabsorption varies considerably between studies and type of feed, with coefficient of fat absorption (CFA) having been reported as varying from 68% to 91% (see for review, Lindquist & Hernell, 2010; Curr Opin Clin Nutr Met Care, 13: 314-320). Several studies have shown that the CFA from heat-treated (pasteurized) human milk is lower than from raw milk Andersson et al, 2007; Acta Paediatr 96: 1445-1449). Furthermore, CFA from infant formulas is lower than from raw human milk given that the fat composition is similar in formula and milk (Chappell et al, 1986). However, since CFA has been reported to decrease with increasing chain length, from C10:0 to C18:0, of a fatty acid (FA) and increases with increasing number of double bonds, C18:0, C18:1 and C18:2 n-6, of the FA (Andersson et al, 2007, reported in, Lindquist & Hernell, 2010), high concentrations of medium-chain triglycerides (MCTs) or of long-chain triglycerides rich in polyunsaturated FA are used in some formulas to increase overall CFA. Of note is that the reported range of CFA, both from human milk and from formulas are wide. This can partly be explained by the amount and composition of fat given, and partly by large interindividual differences in the capacity to utilize dietary fat in preterm newborns, but it also reflects a considerable difficulty in correctly assessing CFA (Hernell, 1999; J Pediatr, 136: 407-409).
Although lipids in human milk and infant formulas are used mainly as an energy substrate, they are also the carrier of indispensible fat-soluble vitamins and provide essential fatty acids of the n-6 and n-3 series; that is linoleic acid (LA) and alpha-linolenic acid (LNA), respectively. Human milk and most formulas intended for preterm infants also provide conditionally essential fatty acids, that is the long-chain polyunsaturated fatty acids (LCPUFAs) derived from LA and LNA, for example arachidonic acid (AA) and docosahexaenoic acid (DHA), respectively.
Some lipids such as cholesterol, phospholipids and LCPUFAs, as constituents of phospholipids, serve as structural components of cell membranes, and the availability and metabolism of them as membrane components directly affect membrane functions. The retina and brain grey matter are particularly rich in LCPUFA, and neural development and functions may depend on their provision by the diet (Uauy & Dangour, 2009; Ann Nutr Metab 55: 76-96; Innis et al, 2009; J Pediatr Gastroenterol Nutr, 48s1: S16-S24], although this has recently been questioned (Beyerlein et al, 2010; J Pediatr Gastroenterol Nutr, 50: 79-85). Certain LCPUFAs regulate gene expression (Jump et al, 2008: Chem Phys Lipids, 153: 3-13) and are precursors of eicosanoids such as prostaglandins, leukotrienes, thromboxanes, and the more recently discovered docosanoids such as resolvins, docosatrienes and neuroprotectins (Serhan et al, 2004; Lipids, 39: 1125-1132; Serhan et al, 2008; Nat Rev Immunol, 8: 349-361). It is therefore evident that both the quantity of dietary lipids used as energy substrate and the quality of dietary structural lipid supply impact on growth, development and function of the newborn infant.
There have been numerous analyses, studies and reviews published that discuss the link between unsaturated fats, especially LCPUFAs, and visual and/or cognitive development or function, for example as summarized by McCann & Ames in 2005 (Am J Clin Nutr, 82: 281-295). Indeed, on the basis of all available evidence, it has been recommended that infant formulas be supplemented with the LCPUFAs docosahexaenoic acid (DHA) and arachidonic acid (AA), and for pregnant and lactating women to include some food sources of DHA in the diet in view of their assumed increase in LCPUFA demand and the relationship between maternal and fetal/infant DHA status (Koletzko et al, 2008; J Perinat Med, 36:36:5-14).
During the last trimester of fetal life and the first 2 years of childhood, the brain undergoes a period of rapid growth termed the “brain growth spurt”. LCPUFAs, particularly DHA and AA, as they are highly concentrated in cell membranes of the retina and brain, accumulate rapidly during the brain growth spurt (Martinez, 1992; J Pediatrics, 120: 129-138). Reduced visual acuity has consistently been observed in primate and rodent offspring subjected to dietary conditions during gestation that result in significant reductions in retinal concentrations of DHA. Human autopsy studies reported significant differences of ˜11% to 40% in DHA concentrations in brain gray matter between breastfed and unsupplemented formula-fed infants (for example, Byard et al, 1995; J Pediatric Child Health 31: 14-16). Direct autopsy evidence that compares brain DHA concentrations in human infants fed unsupplemented and LCPUFA-supplemented formulas is not available. However, an autopsy study in nonhuman primates reported ˜30% lower concentrations of DHA in the visual cortex of preterm infants fed unsupplemented formula than in those fed LCPUFA supplemented formula (Sarkadi-Nagy et al, 2003; Pediatric Res 54: 244-252). In humans, significant differences in plasma concentrations of DHA and AA between unsupplemented and supplemented formula comparison groups are well documented (for example, Boehm et al, 1996; Eur J Pediatr 155: 410-416).
Many experimental studies that investigated the relationship between mental performance and LCPUFAs have been conducted using rodents. Many early studies suggested an association between a diet severely restricted in n-3 fatty acids during development and poorer performance of offspring in tests designed to measure cognitive or behavioral ability (for example, reviewed in Wainright, 1992; Neurosci Behav Res 16: 193-205). Furthermore, McCann & Ames reviewed eight studies that supplemented n-3-restricted animals with DHA, DHA+AA, DHA-rich oils, or DHA and additional n-6 fatty acids and compared the animals' performance with that of n-3-restricted controls. All of these studies reported that performance was significantly enhanced in the supplemented groups.
McCann & Ames also considered five systematic reviews published since 1999 that critically evaluated partially overlapping subsets of breastfeeding studies spanning over 20 years. Most of the studies included in these reviews compared the performance of children who were breastfed or formula-fed. Before adjustment for covariables, most of these studies reported higher scores on performance tests for children who were breastfed.
Information on the question of causality provided by observational breastfeeding studies, although relevant, is limited. Randomized controlled trials offer much greater opportunity than do observational studies for the control of experimental variables, including the quantity and composition of LCPUFA supplements. In addition, this design affords the opportunity to avoid many of the potential confounding factors that complicate the interpretation of observational breastfeeding studies. In a randomized clinical trial, Willatts and coworkers (1998; The Lancet, 352: 688-691) observed that infants who received LCPUFA-supplemented formula until age 4 months had significantly more intentional solutions when tested at age 10 months than those who received unsupplemented formula. Based on these results, the authors suggested that infants may benefit from LCPUFA supplementation and that the effects persist beyond the period of supplementation. Furthermore, since higher-problem-solving scores in infancy are related to higher IQ scores, they speculated that supplementation may be important for the development of childhood intelligence. In another randomized clinical trial (Birch et al, 2000; Devel Med Child Neurol, 42: 174-181), supplementation of infant formula with DHA+AA was associated with a mean increase of 7 points on the Mental Development Index (MDI) of the Bayley Scales of Infant Development, 2nd edition test (BSID-II). Both the cognitive and motor subscales of the MDI showed a significant developmental age advantage for DHA− and DHA+AA-supplemented groups over the control group. While a similar trend was found for the language subscale, it did not reach statistical significance. Significant correlations between plasma and red blood cell-DHA at 4 months of age but not at 12 months of age and MDI at 18 months of age suggest that early dietary supply of DHA was a major dietary determinant of improved performance on the MDI. Recently however, a meta-analysis of 4 large clinical trials showed no effect on infant development, as assessed by the Bayley test at 18 months, of formulae supplemented with DHA as compared to unsupplemented formulae (Bayerlein et al, 2010).
A normal human pregnancy lasts for about 40 weeks (38-42 weeks), and the WHO defines prematurity as a baby born before 37 full-weeks from the first day of the last menstrual period. Premature babies are susceptible to a number of health problems and many require specialized care in Newborn Intensive Care Units (NICUs). Of particular significance however, has been the suggestion that normal uterine growth may be very important in terms of early growth of the brain, and premature birth may lead to poor IQ and developmental skills (Cook, 2006; Arch Dis Child Fetal Neonatal Ed, 91: 17-20). Individuals who were born before 33 weeks gestation continue to show noticeable decrements in brain volumes and striking increases in lateral ventricular volume into adolescence (Nosarti et al, 2002; Brain, 125: 1616-1623). Whether such neurological changes are causative remains disputed. However, during follow-up in school life, it has been seen that cognitive and neuromotor impairments at 5 years of age increase with decreasing gestational age. Many of these children need a high level of specialised care (Larroque et al, 2008; Lancet, 8; 371: 813-820). In particular, about half of infants born at 24-28 weeks of gestation have such a disability at 5 years, and in the infants born later (29-32 weeks' gestation), about a third have such a disability at 5 years (Marlow et al, 2005; N Engl J Med, 352: 9-19). Furthermore, other studies have seen association between gestational birth age and behavioral and psychomotor problems. For example, a study from Liverpool (UK) has looked at children of age 7 and 8 who were born before 32 weeks and who were well enough to attend mainstream school, compared with full-term children of similar postpartum age in their class at school (Foulder-Hughes & Cooke, 2003; Dev Med Child Neurol, 45: 97-103). This study suggested that: (i) the preterm children had a higher incidence of motor impairment and this affected how well they did at school even when their intelligence was normal; (ii) over 30% had developmental coordination disorder (DCD) compared with 6% of classmates; (iii) the preterm children were significantly more likely be overactive, easily distractible, impulsive, disorganized and lacking in persistence. They also tended to overestimate their ability; (iv) attention deficit hyperactivity disorder (ADHD) was found in 8.9% of the preterm children and only 2% of controls. Of note however, was that the children who had been the most premature were not necessarily those with the lowest scores and, in comparison to historical studies, although major disabilities have been reduced, the levels of those disabilities tested in this study did not seem lower than those found in children born 10 or 20 years earlier, despite improvements in care of the newborn.
As described above, pancreas and liver functions are not fully developed at birth, and in premature infants this is particularly notable. Lindquist and Hernell (1990; Curr Opin Clin Nutr Metab Care, 13: 314-320) have recently reviewed the subject of lipid digestion and absorption in early life. Breast-fed infants digest and absorb fat (and importantly LCPUFAs) more efficiently than formula-fed infants (Bernback et al, 1990; J Clin Invest, 85:1221-1226; Carnielli et al, 1998). In addition to infant formulas of similar fat composition, mother's milk also contains a broad-specificity lipase, bile-salt stimulate lipase (BSSL) (EC 3.1.1.13) that promotes highly efficient fat absorption from human milk.
BSSL is believed to have a broader substrate specificity than most lipases. Not only is the enzyme capable of completely hydrolyzing all three fatty acids of TG, but also fat soluble vitamin esters such as vitamin A as well as cholesteryl esters. Thus, BSSL drives the intraluminal lipolysis toward completion and results in the formation of glycerol and free fatty acids (FFAs), including long-chain polyunsaturated fatty acids (Hernell, 1975; Eur J Clin Invest, 5: 267-272; Bernback et al, 1990; Hernell et al, 1993; J Pediat Gastro Nutr, 16: 426-431; Chen et al, 1994; Biochem Biophys Acta, 1210: 239-243). BSSL shows optimal activity at a pH of 8-8.5 and is more stable in acid environments than pancreatic lipase. BSSL is resistant to degradation by pepsin at physiological concentrations. BSSL accounts for about 1% of the total protein in milk and is present at concentrations from 0.1-0.2 g/L (Blackberg et al, 1987; FEBS Lett, 217: 37-41; Wang & Johnson, 1983; Anal Biochem, 133: 457-461; Stromqvist et al, 1997; Arch Biochem Biophys, 347: 30-36). The levels of BSSL in human milk are similar throughout the day (Freed et al, 1986; J Pediatr Gastroenterol Nutr, 5: 938-942) and BSSL production in human milk is maintained for at least 3 months (Hernell et al, 1977; Am J Clin Nutr, 30: 508-511) although concentrations of BSSL may decline with duration of lactation (Torres et al, 2001; J Natl Med Assoc, 93: 201-207). Triglycerides comprise about 98% or more of all lipids in human milk or formula and thereby account for about 50% of the energy content.
Using fresh human milk as a (realistically) complex source of TGs and BSSL, Hall & Muller (1982; Pediatr Res 16: 251-255) concluded that BSSL showed little specificity for different fatty acids of TG. In contrast, using (an artificial system of) an equimolar mixture of monoacid TGs, Wang & coworkers (1983; J Biol Chem, 259: 9197-9202) suggested that BSSL hydrolyzed the short chain TGs more readily than long-chain, and that C18:2 fatty acids were hydrolyzed faster than C18:1 and C18:0. Jensen & coworkers (1985; J Pediatr Gastroenterol Nutr, 4: 580-582) obtained evidence of biased hydrolysis of an asymmetric TG in favor of hydrolysis of C18:2 fatty acid. Using radiolabeled rat-derived chylomicrons, Hernell and coworkrs (1993; J Pediatr Gastroenterol Nutr, 16: 426-431) concluded that BSSL did not differentiate between the hydrolysis of LA (C18:2 n-6) or AA (C20:4 n-6) or between that of AA and eicosapentaenoic acid (C20:5 n-3). In a similar assay, Chen and coworkers (1994; Biochim et Biochphys Acta, 1210: 239-243) obtained evidence that BSSL hydrolyzed DHA fatty acids (C22:6 n-3), but less efficiently than C18.1 or AA, and speculated that BSSL may have a physiological role in completing duodenal hydrolysis of milk TG containing DHA or AA esters to free fatty acids and glycerol.
The superiority of human milk as a nutritional source for term as well as preterm infants has been manifested in many studies and expert group recommendations. Accordingly, the recommended feeding method world-wide is breastfeeding. Neither is however, breastfeeding nor feeding the mother's own breast milk always possible or recommended for medical reasons—and breastfeeding may not be practiced for a number of other reasons—in each case as discussed elsewhere herein. In cases where the infant is not breast-fed, infant formula or banked and non-banked pasteurized and/or frozen breast milk is often used. All are, however, in some respects nutritionally suboptimal for newborn infants.
Due to risks of viral infection (human immunodeficiency virus [HIV], cytomegalovirus [CMV], hepatitis) and to a lesser degree transmission of pathogenic bacteria, donor milk used in so-called milk banks is generally pasteurized before it is used. However, BSSL is inactivated during pasteurization of human milk (Björksten et al, 1980; Br Med J, 201: 267-272); nor is it present in any of the many different formulas that exist for the nutrition of pre- or full-term neonates. It has been shown that fat absorption, weight gain and linear growth is higher in infants fed fresh compared to pasteurized breast milk (Andersson et al. 2007; Williams et al, 1978; Arch Dis Child 43: 555-563). This is one reason why it has been advocated that newborn infants, particularly preterm infants, that cannot be fed their own mothers milk should be fed non-pasteurized milk from other mothers (Bjorksten et al, 1980).
Hamosh (1983; J Ped Gastro Nutr, 2: 248-251) reported that BSSL enzyme activity is present in fresh breast milk of women who delivered at 26 to 30 weeks. This report further described that milk specimens stored at −20 or −10° C. showed a slow loss in BSSL activity, but a more dramatic loss of bile-salt dependency on activity after only three weeks storage at −10° C. which may contribute to hydrolysis of milk lipids even during storage of breast milk at −20° C.
Milk bile-salt-stimulated lipase has been found only in the milk of certain species, namely humans, gorillas, cats and dogs (Freed, et al, 1986; Biochim Biophys Acta, 878: 209-215). Milk bile-salt-stimulated lipase is not produced by cows, horses, rats, rabbits, goats, pigs or Rhesus monkeys (Blackberg et al, 1980; Freudenberg, 1966; Experientia, 22: 317).
Native human milk BSSL (hBSSL-MAM) has been purified to homogeneity, as reported by Blackberg and Bernell (1981; Eur J Biochem, 116: 221-225) and Wang & Johnson (1983), and the cDNA sequence of human BSSL was identified by Nilsson (1990; Eur J Biochem, 192: 543-550) and disclosed in WO 91/15234 and WO 91/18923. Characterization and sequence studies from several laboratories concluded that the proteins hBSSL-MAM and the pancreas carboxylic ester hydrolase (CEH) (also known as pancreatic BSSL) are both products of the same gene (for example, Baba et al, 1991; Biochem, 30: 500-510 Hui et al, 1990; FEBS Lett, 276: 131-134; Reue et al, 1991; J Lipid Res, 32: 267-276).
Following the isolation of the cDNA sequence, recombinant human BSSL (rhBSSL), as well as variants thereof, has been produced including in transgenic sheep (rhBSSL-OVI); such as described in U.S. Pat. No. 5,716,817, WO 94/20610 and WO 99/54443. Production of proteins for therapeutic use using transgenic animals has been met with significant safety, scientific, regulatory and ethical resistance. Indeed, to date there is no approved therapeutic product on the US or EU market that has been produced from transgenic sheep, and only two medical products produced from other transgenic animals have so far been approved: ATRYN (recombinant antithrombin) produced from transgenic goats, and RUCONEST (recombinant component 1 esterase inhibitor) produced from transgenic rabbits. Proteins produced in such a manner (to be expressed in mammary tissue and excreted in milk) can be contaminated with components naturally found in the milk of these animals, such as whey or non-human milk or whey proteins, which may cause safety issues if such proteins are used for human use in certain individuals, such as those intolerant or allergic to milk-based components or products.
It has long been promoted that fresh human breast milk is the most suitable feed for human infants. This is based on studies such as the early work by Williams et al (1978) who showed that heat-treatment of human milk reduced fat absorption by approximately one-third (compared to raw human milk) in an experimental study of seven VLBW preterm infants (less than 1.3 Kg) aged between 3 and 6 weeks, fed for three consecutive weeks with raw, pasteurized and boiled human milk, each for one week. This study made the suggestion that the improvement in fat absorption may be related to the preservation of milk lipases in the raw, compared to the heat-treated, human milk. Of note is that this study described that all infants gained weight most rapidly during the week in which they were fed raw milk; with the mean weight gain (reported in g gained per week per 100 mL milk consumed) during this period approximately one third greater than the similar periods during which pasteurized or boiled milk was administered. In a larger (but shorter) study reported by Alemi (1980; Pediatrics. 68: 484-489), fat excretion was studied in 15 VLBW infants, born with a birth-weight of between 660 and 1,695 g and a gestational age of 26 to 33 weeks, and the study started at 7 to 44 days after birth. Fat excretion was lower in those infants fed a mixture of human milk and formula for 72 hours compared to the infants fed formula only. More recently, Andersson & coworkers (2007) reported in a randomized study that pasteurization of mother's own milk reduced fat absorption and growth in preterm infants, and proposed that these effects were due to inactivation of milk-based BSSL by pasteurization. Of note is that the reported range of coefficient of fat absorption (CFA) from a number of studies, including those above, are wide; both from human milk and from formulas. This can partly be explained by the amount and composition of fat given, and partly by large interindividual differences in the capacity to utilize dietary fat in preterm newborns, but it also reflects a considerable difficulty in correctly assessing CFA (Hernell, 1999; J Pediatr, 136: 407-409).
One animal model study has attempted to investigate the effects on infant growth by the addition on exogenous BSSL to neonatal food (Wang et al, 1989; Am J Clin Nutr, 49: 457-463. This study involved the addition of purified human BSSL (0.1 mg/mL) to kitten-formula (mixed three to one with cow milk) and then fed to to six bottle-fed kittens for 5 days. This study reported that kittens fed with kitten-formula supplemented with hBSSL had a growth rate of twice that of those fed with formula alone. Of note is that the formula was supplemented with cow milk, the kittens were not preterm or of low birth weight, they were breast fed for the first 48 hours of their life and the study was conducted with purified native hBSSL. The authors suggested that the kitten could be utilized as an animal model in the investigation of the functional role of BSSL, and on the basis of this study related patent applications were filed (including, U.S. Pat. No. 4,944,944, EP 0317355 and EP 0605913) that disclose (amongst other aspects): a method for fortifying a fat-containing infant formula which is poor in bile-salt-activated lipase comprising adding to the formula an effective amount of an isolated bile-salt-activated lipase selected from the group consisting of milk bile-salt-activated lipase [BSSL] and bile-salt-activated pancreatic carboxylesterase [now known to also be BSSL] to increase fat absorption from the formula and growth of the infant; and a method for feeding an infant a dietary base from a first source comprising fats consisting of administering an isolated bile-salt-activated lipase selected from the group consisting of milk bile-salt-activated lipase [BSSL] and bile-salt-activated pancreatic carboxylesterase [also BSSL] to the infant in an amount sufficient to improve the infant's digestion and absorption of the fats in the base and increase the growth of the infant, wherein the lipase is derived from a second source. No data supporting an improvement in fat absorption were disclosed, not any data obtained from any study that involved human infants. Another study (Lindquist et al, 2007; J Pediatr Gastroenterol Nutr 44: E335) has been reported by Lindquist & Hernell (2010) as artificially feeding purified human BSSL to BSSL-knock-out mice pups nursed by BSSL-knock-out dams to restore normal fat absorption and preventing the formation of intestinal lesions.
Following the cloning of the hBSSL cDNA and the disclosure of various approaches to produce large quantities of recombinant human BSSL (rhBSSL), numerous disclosures have been made, and claims to, various infant formulas comprising rhBSSL (for example, U.S. Pat. No. 5,200,183, WO 91/15234, WO 91/18923, and U.S. Pat. No. 5,716,817) and various methods or uses of such formula or rhBSSL, including as an infant supplement, for the improvement of dietary lipids, treatment of fat malabsorption, certain pancreatic abnormalities and cystic fibrosis (for example, WO 91/18923, WO 94/20610 and WO 99/54443). However, as with the earlier suggestive studies, no supporting data obtained from experiments supplementing human infants with recombinant bile-salt-stimulated lipase are disclosed. Indeed, in 1996 after all these suggestions, associative studies and disclosures, leading workers in the area were still questioning: “Should bioactive components of human milk [such as BSSL] be supplemented to formula-fed infants?”; and further stating that: “There are no data on attempts to supplement digestive enzymes [such as BSSL]” (Hamosh, at Symposium: Bioactive Components in Milk and Development of the Neonate: Does Their Absence Make a Difference? Reported 1997, in J Nutr, 12: 971-974). More recently, Andersson and coworkers (2007) have speculated that supplementing pasteurized milk with recombinant human milk BSSL may restore endogenous lipolytic activity of the milk.
The 722 amino-acid native BSSL is heavily glycosylated (30-40% carbohydrate) (Abouakil et al, 1989; Biochem Biophys Acta, 1002: 225-230), with extensive O-glycosylation sites within the C-terminal portion of the molecule that in its most abundant form contains 16 proline-rich repeats of 11 residues with O-linked carbohydrates (Hansson et al, 1993; J Biol Chem, 268: 26692-26698). The role of the extensive O-glycosylation is unproven, but based on its sequence composition the large C-terminal tail is predicted to be very hydrophilic and accessible (Wang et al, 1995; Biochemistry, 34: 10639-10644).
Differences in glycosylation patterns can have dramatic differences in the activity or other properties of many proteins, especially proteins used in medicine. For example, ARANESP (darbepoetin alpha) is a specifically engineered variant of erythropoietin that differs from PROCRIT (epoetin alpha) by 2 amino acids that provides the molecule with 5 N-linked oligosaccharide chains rather than 3, and which significantly alter the pharmacokinetic properties; with darbepoetin showing a threefold increase in serum half-life and increased in vivo activity compared to epoetin (Sinclair and Elliot, 2005; J Pharm Sci 94: 1626-1635).
Different recombinant production systems (such as mammalian cell, yeast, transgenic animal), and even seemingly minor changes in production process from the same expression system, can lead to changes in the glycosylation of the same protein/polypeptide sequence. For example, recombinant human alpha-galactosidase A is used in enzyme replacement therapy for Fabry's disease, and the commercial drug product is produced in two ways, having the same amino acid sequence but each having a different glycosylation pattern: REPLAGAL (agalsidase alfa) and FABRAZYME (agalsidase beta). REPLAGAL is produced in a continuous line of human fibroblasts while FABRAZYME produced in Chinese hamster ovary (CHO) cells, and each product has different glycosylation. In common with other proteins produced from CHO cells, FABRAZYME is a sialyated glycoprotein, and has differences in the degree of sialyation and phosphorylation compared to REPLAGAL (Lee et al, 2003; Glycobiology, 13: 305-313). The qualitative and quantitative differences in the sialylation of glycoproteins produced in CHO cells in comparison with natural human glycoproteins have consequences for both the level of biodistribution and immunogenic potency. In fact, the presence of IgG has been reported in almost all patients treated with agalsidase beta compared to only 55% of patients treated with agalsidase alfa (Linthorst et al, 2004; Kidney Int, 66: 1589-1595). Moreover, in some cases, an allergic type reaction to treatment with agalsidase beta has been recorded, with the presence of IgE in the circulation and/or a positive intradermal reaction (Wilcox et al, 2004; Am J Hum Genet, 75: 65-74).
Indeed, while their peptide maps are very similar, the glycosylation patterns of native BSSL does differ substantially from that of rhBSSL produced in mouse C127 and hamster CHO cell lines, and also in the ability to bind to certain lectins including concanavalin, Ricinus communis agglutinin and Aleuria aurantia agglutinin suggesting that native BSSL contains considerably more fucose and terminal beta-galactose residues than the recombinant forms (Stromqvist et al, 1995; J Chromatogr, 718: 53-58). Landberg et al (1997; Arch Biochem Biophys 344: 94-102) further characterized these two recombinant forms, and reported that both recombinant forms had a lower molar percent of total monosaccharide (20% and 15% for C127- and CHO-produced rhBSSL, respectively, compared to 23% for native hBSSL), and that while native hBSSL reacted to certain Lewis antigen-detecting antibodies, the C127-rhBSSL did not.
Although the C127- and CHO-produced rhBSSL reported above were generally similar to each other in terms of molecular mass, glycosylation and lectin binding, in contrast, the rhBSSL isolated from the milk of transgenic mice showed a lower apparent molecular mass on size-exclusion chromatography (SEC) and no detectable interactions with a panel of lectins, indicating a significantly lower degree of O-glycosylation of rhBSSL in milk from transgenic mice than found for the other recombinant forms (Stromqvist et al, 1996; Transgen Res 5: 475-485).
Clinical studies in specific indications conducted with one particular form of rhBSSL have been reported; namely early-phase exploratory studies of exocrine pancreatic insufficiency (PI) due to chronic pancreatitis or cystic fibrosis (CF). In 2004, a phase II trial was reported that showed that CF patients (aged 12 to 39 years) with PI had a more rapid and efficient lipid uptake when supplemented with rhBSSL at a single dosing of 0.2 g or 1 g as a complement to 25% of their regular Creon dosing, as compared to Creon alone given at their regular does, or at 25% dosage (Strandvik et al, 2004; 18th North American Cystic Fibrosis Conference, St Louis M I; abstract published in Pediatr Pulmonol, S27: 333), and in 2005 the results of a second phase II trial were reported as rhBBSL showing a greatly improved ability of a group of Swedish patients with CF suffering from PI to digest fat (press release from Biovitrum, reporting Strandvik et al, 2005; 28th European Cystic Fibrosis Society (ECFS) Conference, Crete). In both clinical trials, these clinical results were obtained using rhBSSL-OVI. More recently, it has been announced that a further phase II trial with an oral suspension of rhBSSL (described therein as “bucelipase alpha”), dosed at 170 mg 3 times daily for 5-6 days, to evaluate the effect on the fat absorption in adult patients with CF and PI has been completed, but no efficacy results from this have to date been published (clinicaltrials.gov identifier NCT00743483).
It has been disclosed since at least 2008 that two phase II trials using rhBSSL were planned and ongoing, each to investigate the coefficient of fat absorption, and change in length and body weight, in preterm infants born before 32 weeks gestational age treated with 0.15 g/L rhBSSL or placebo for one week each, added to infant formula (clinicaltrials.gov identifier NCT00658905) or to pasteurized breast milk (clinicaltrials.gov identifier NCT00659243).
In light of the prior art, and the long felt need for a solution, it is therefore an object of the present invention to provide a method of increasing the absorption of at least one unsaturated fatty acids, such as essential fatty acids or LCPUFAs, by a human infant, such as a preterm human infant. Said method should overcome one or more of the disadvantages of the prior art, that include: that an active ingredient that can be reliably and/or reproducibly produced in large quantities; that the active ingredient has been manufactured by a scientifically, regulatory and/or ethically acceptable method; and/or that the method or the active ingredient used in the method, has been demonstrated, within a randomized clinical trial involving human infants, to be efficacious and safe.
The solution to the above technical problem is provided by the various aspects and embodiments of the present invention as defined or otherwise disclosed herein and/or in the claims.